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Highlights:» Established efficient method to produce “isogenic” iPS cell lines» Developed series of assay tools involving GPCRs» Created models of sudden death syndrome

Genome EngineeringThe late Richard Feynman once said, “What I cannot create, I do not understand.” Human genetics began as observational science, but newly developed genome-engineering tools now allow us to directly test the cellular consequences of discrete genetic changes. We have developed efficient methods to edit one residue at a time in living human induced pluripotent stem (iPS) cells, resulting in “isogenic” lines of iPS cells. These isogenic lines form models that are now yielding phenotypes that are helping to explain the molecular basis of several human diseases. We are constructing collections of these isogenic lines that carry a range of disease mutations, from the most severe (rare) to the moderate (common) forms of cardiomyopathy.

Precise Genome Editing for Human TherapyThe rapid development of genome engineering, such as with the CRISPR system, now allows us to contemplate using these tools for human therapy in selective tissues. Using genome editing to “fix” a disease gene has great promise, because it could permanently correct the disease. However, tremendous challenges remain to ensure that only a specific part of the genome is edited, without causing “off-target” DNA damage that could lead to cancer. We focused on developing therapeutic editing for diseases of the heart and retina, because each tissue has unique challenges and opportunities. The heart has many newly described gene mutations that result in lethal heart failure (cardiomyopathy). Genome engineering can be used to cure cardiomyopathy mutations and validate targets for drug therapy; however, current engineering methods will need to be greatly enhanced before it can be used in vivo in the heart. In contrast, the retina has the advantage of a limited number of cells that can be directly targeted for therapy. These properties could potentially cure blindness caused by many gene mutations that currently have no therapy. We are developing proof-of-concept therapeutic editing methods in the retina that can be expanded to other tissues, such as the heart.

Human Cardiac Disease ModelsWe use iPS cells to model the genetic causes of human cardiomyopathy. Surprisingly, little is known about many genes associated with heart failure, from cardiomyopathy to abnormal heart rhythm, that results in “sudden death.” The heart provides an ideal system to determine the molecular basis of gene associations in human genetics. Until recently, modeling human gene variants in human cardiac tissue has been impossible. Human iPS cells now allow us to produce cardiovascular tissues that are identical, except for a single gene that has been altered by genome engineering. Our efforts have already been used to uncover multiple disease phenotypes and targets for drug therapy.

CRISPR-Based Screens in Human Cardiac Disease ModelsRecently, we developed CRISPR-inhibition (CRISPRi) cell lines for highthroughput gene inactivation of thousands of different genes. CRISPRi screens can be used to identify the molecular basis of development, so that we can construct more mature human tissues and improved disease models. These studies will also allow us to identify drug targets that could be used for treating cardiomyopathy and other major diseases. We are currently focused on finding new pathways to enhance cardiac regeneration. In the future, other versions of the CRISPR system could be used to activate genes and control the epigenetic state of the genome.

Future DirectionsWe are developing new genome engineering methods in human iPS cells to identify therapeutic targets in cardiac disease. We are using these same tools to develop strategies for therapeutic genome editing. The combination of human iPS cells and genome editing provide unprecedented opportunities to explore new areas of biology and discover new therapies for disease.